13C glucose breath test for the diagnosis of diabetic indications and monitoring glycemic control

ABSTRACT

A breath test and kit for performing the breath test are described for the diagnosis of diabetic indications and monitoring of glycemic control. The breath test utilizes the measurement of expired  13 C-labeled CO 2  following the ingestion of a  13 C-enriched glucose source.

REFERENCE TO RELATED APPLICATION

[0001] This application claims priority on U.S. provisional applicationNo. 60/084,482 filed May 6, 1998, which is relied upon and incorporatedherein by reference.

BACKGROUND OF THE INVENTION:

[0002] Glucose tolerance is defined as the ability to properly utilizeglucose. Diabetes is not a single disease, but an array of diseases thatexhibit the common symptom of glucose intolerance, an impairment inglucose utilization.

[0003] The prevalence of diabetes in the general population isapproximately 6-7%. Only about half of diabetics are actually diagnosed.Studies have shown that rates for persons with glucose intolerance areequal by sex and greater for blacks than for whites.

[0004] In general, the following types of diabetes have been recognized,type I diabetes mellitus, type II diabetes mellitus, secondary diabetesmellitus, impaired glucose tolerance and gestational glucose mellitus.The general characteristics of the symptoms of diabetes include thefollowing:

[0005] Polyuria (high urine blood volume)

[0006] Hyperglycemia (high blood glucose levels)

[0007] Glucosuria (loss of glucose in urine)

[0008] Polydipsia (excessive thirst)

[0009] Polyphagia (excessive hunger)

[0010] Sudden weight loss

[0011] It has been observed that complications resulting from diabetesmellitus are the third leading cause of death in most developedcountries. Diabetes is a risk factor for a variety of conditionsincluding coronary heart disease, cerebrovascular stroke, neuropathy(nerve damage), nephropathy (kidney damage), retinopathy (eye damage),hyperlipidemia (excessive blood lipids), angiopathy (damage to bloodvessels) and infection.

[0012] A number of different methods exist for determining a conditionof intolerance for glucose. These include postprandial blood glucose,oral glucose tolerance test (OGTT), O'Sullivan glucose tolerance test(gestational test), hemoglobin Alc (Hb A_(l), Hb A_(lc)), islet cellantibodies, GAD antibodies (glutamic acid decarboxylase) and insulinantibodies. Diabetes, however, is most readily detected when thecarbohydrate metabolic capacity is tested. This is done by stressing thesystem with a defined glucose load as in the oral glucose tolerance test(OGTT).

[0013] The OGTT has been criticized, however, because many of thevariables affecting test results are difficult to control, for instance:Patients must be on a standardized carbohydrate diet at least three daysbefore the test. The test requires an 8 to 16 hour fast. The test shouldonly be performed on ambulatory patients. Stress should be avoided.Exercise should be avoided. Various hormone imbalances can affectvalidity such as with: thyroxine, growth hormone, cortisol andcatecholamines. Various drugs and medications can affect validity suchas: oral contraceptives, salicylates, nicotinic acid, diuretics andhypoglycemics. Evaluation should normally be corrected for age. Thegreatest disadvantage of the OGTT is that it is poorly reproducible andthis limits its diagnostic usefulness.

[0014] The current methods of diagnosing diabetes involve eitherinvasive testing (ie. repeated blood collections), or use blood-bornemarkers (ie. glycosylated proteins, or antibodies) which offer anindirect assessment of glucose regulation. Accordingly, it is an objectof the present invention to avoid the need for invasive testing or theuse of blood-borne markers in determinations of glucose regulation.

SUMMARY OF THE INVENTION

[0015] The above and other objects of the invention are attained by a¹³C breath test and a kit for determining glucose regulation in apatient in need thereof.

[0016] An analytical assay is described that is based on the use ofnon-radioactive ¹³C. Labeled expired ¹³CO₂ is measured in the presentassay. Isotope ratio mass spectroscopy (IRMS) is used as a detectionmethod for ¹³C, a non-radioactive isotope that occurs naturally in foodand animal tissues. Non-dispersive infrared spectroscopy (NDIRS)analysis and analysis methods known in the art may be employed. The testprotocol is as follows: after an overnight fast, the oral dose of ¹³Cuniformly labeled glucose (containing about 25 mg of ¹³C glucose incombination with about 15 g of unlabeled glucose in 100 mL of tap water)is administered. Breath samples will be collected before the dose andthen 1½ hours after ¹³C glucose ingestion. Levels of ¹³CO₂ in expiredair will be measured by an IRMS method.

[0017] Advantages of this test are the following:

[0018] it is practical, sensitive and specific;

[0019] the validity of the test is not influenced by stress, exercise,hormone imbalances, or some drugs and medications

[0020] it is a non-invasive method;

[0021] it is simple to perform and can be readily used in physicians'offices or medical laboratories;

[0022] it is safe since ¹³C is a naturally occurring isotope found inall carbon-containing substances;

[0023] it involves no radioactivity, and may be used in children andwomen.

[0024] The ¹³C glucose test is safe, reliable, and specific in diagnosisof diabetes and measurement of the severity of insulin resistance inpatients. The invention is also preferred to diagnose gestationaldiabetes and to monitor glycemic control in diabetes patients. Aprefered embodiment of the invention is a kit containing the necessarymaterial for performing the described method. This kit may contain butis not limited to a source of ¹³C enriched glucose (preferably uniformlylabeled D-glucose); a source of unenriched glucose; and a breathcollection device. The kit may also contain a set of patientinstructions for its use. In another embodiment, the kit mayadditionally contain a blood collection device such as a lancet orhypodermic needle and vacutainer for the additional determination ofblood glucose levels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1: Illustrates the IRMS analysis of ¹³C glucose breathsamples from normal individuals, a gestational diabetic and patientswith impaired glucose tolerance.

[0026]FIG. 2: Shows a representative example of breath test and bloodglucose levels of a normal individual.

[0027]FIG. 3: Illustrates breath test and blood glucose levels of adiabetic patient.

[0028]FIG. 4: Depicts breath test and blood glucose levels of an insulinresistant patient.

[0029]FIG. 5: Shows a comparison of IRMS results of an insulin resistantand a diabetic patient and a normal individual.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The introduction of a ¹³C breath test offers a novel,non-invasive, direct means to monitor glucose metabolism by measurementof exhaled CO₂ using highly enriched, uniformly labeled ¹³C-glucose.Glucose metabolism will generate labeled CO₂, which is then exhaled andcollected in tubes. Enrichment of labeled CO₂, over a determined timecourse, can be used as a quantitative index of glucose metabolism.Comparison is made against age-specific reference intervals.

[0031] The present invention has a number of advantages, including lowerdose of glucose needed (overcomes inconsistencies due to malabsorptivedisorders or previous gastric or intestinal surgery), reduction intesting time (from the current 2 hours required for the OGTT) and fewerinterpretational ambiguities (greater sensitivity and specificity).

[0032] The ¹³C glucose breath test is based on the metabolism ofglucose. Following a baseline breath sample, a ¹³C glucose solutioncontaining about 25 mg of ¹³C glucose in combination with about 15 g ofunlabeled glucose in 100 mL of tap water is administered. Breath sampleswill be obtained before the dose and then 12 hours after ¹³C glucoseingestion. Measurement of the expired air will be detected by an isotoperatio mass spectroscopy assay method. Elevated or excessive breath 13CO2concentrations will be seen in individuals who have normal glucosemetabolism.

[0033] The following Examples serve to illustrate the present invention.These Examples are not intended to limit the scope of the invention inany manner.

EXAMPLE 1 Sample Assay for Diagnosis of a Patient

[0034] Experimental Procedure

[0035] Medical History

[0036] Medical history is taken and includes, but is not limited to: theabsence of active pulmonary disease, no history of heart, liver, orrenal failure, and no use of insulin or oral medications for thetreatment of diabetes.

[0037] Physical Examination and Laboratory Tests

[0038] No physical examination or laboratory tests, including bloodsampling, is required.

[0039] Dietary Control

[0040] It is determined that all participants have fasted overnightprior to commencement of the test.

[0041] Patient Control

[0042] Participants are not permitted to eat, drink, or smoke during thetest. All patients are required to remain sedentary for the duration ofthe test. Small amounts of water are allowed.

[0043] Assay Procedure

[0044] Patients fast for at least 8 hours before this test.

[0045] A sample set of patient instructions is given below:

[0046] Step 1: COLLECT FIRST BREATH SAMPLE

[0047] Remove the screw cap from the collection tube.

[0048] Take a normal breath and then exhale fully 4 to 8 seconds througha straw into the bottom of the collection tube.

[0049] Immediately replace the screw cap on the collection tube andtighten until snug (do not overtighten).

[0050] Affix the completed green label to the collection tube.

[0051] Step 2: DRINK THE SOLUTION

[0052] Prepare the solution by adding tap water to the fill line on theplastic container. Mix until completely dissolved and then drink theentire solution.

[0053] Wait 1½ hours.

[0054] Step 3: COLLECT THE SECOND BREATH SAMPLE

[0055] One and one half hours after drinking the solution, collect thesecond breath sample into the collection tube following the samedirections as for the first breath sample in step 1.

[0056] Affix the completed yellow label to the tube.

[0057] Step 4: RETURN THE SAMPLES FOR ANALYSIS

[0058] Insert the 2 collection tubes along with the signed and completedregistration card in the mailing box.

[0059] Return the mailing box as instructed to the site of dispensing.

EXAMPLE 2 Breath Test Administration

[0060] Patients are given an exetainer tube with the screw cap removed.Using the straw, they are asked to breathe into the tube, exhalingnormally, for 4 to 8 seconds. Next, each patient is instructed to drinka solution containing about 25 mg of uniformly labeled ¹³C glucose incombination with about 15 g of unlabeled glucose in 100 mL of tap water.After 12 hours, the patients are given a new tube to breathe in asdescribed above. The breath collection is then complete.

[0061] Storage and Shipping

[0062] Breath test tubes are typically labeled with the patient's nameand identification number and shipped to an analytical laboratory foranalysis. No refrigeration or special storage techniques are necessary.

EXAMPLE 3 Analytical Methodology

[0063] Breath specimens are analyzed by isotope ratio mass spectroscopy.NDIRS is also a preferred method to analyze breath test samples. Othermethods known in the art may also be used.

[0064] Statistical Analysis

[0065] The sensitivity, specificity, positive and negative predictivevalues of the breath test are compared to that of the oral glucosetolerance test. Receiver operator characteristic curve analysis isperformed to confirm the discrimination between type 2 diabetes orgestational diabetes and individuals with normal glucose metabolism.

EXAMPLE 4 Basis of the Method of IRMS

[0066] Isotope ratio mass spectroscopy (IRMS) is a highly precise methodof analysis which is able to measure small samples (low nanogramamounts). For example, ¹³C/¹²C ratios are determined on a mono-carbonmolecule; CO₂ gas. The CO₂ gas can be directed to the spectrometer bymeans of a continuous flow IRMS (also called CF-IRMS).

[0067] The statistical combination of the isotopes of carbon (¹²C and¹³C) and oxygen (¹⁶O, ¹⁷O, ¹⁸O) to generate the CO₂ molecules gives riseto the formation of various isotopomers whose molecular weights are 44,45, and 46, respectively. Thus, for measuring carbon isotope ratios,three ion beams are generated and recorded in the IRMS, corresponding tothe masses of the various isotopomers of CO₂.

[0068] In order to obtain a high precision and a high accuracy,reference gases of absolutely known isotopic composition are used and adual inlet system allows an alternative admission of both sample andreference gases into the ionization source via a gas-switching valve.The measurement of the various ion beams allows for the calculation ofthe ¹³C enrichment of the sample. The value of this calculation is givenδ¹³C({fraction (0/00)}) notation. The ¹³C abundance is expressed asδ¹³C({fraction (0/00)}) according to the following:

δ¹³C({fraction (0/00)})=([(¹³C/¹²C) sample/(¹³C/¹²C)PDB]−1)×1000

[0069] This δ¹³C({fraction (0/00)}) value measures the variations inparts per thousand of the carbon isotope ratio from the standard. Forcarbon, PDB was selected as the international reference. PDB is Pee DeeBelemnitella (a fossil from the Pee Dee geological formation in SouthCarolina). The ¹³C/¹²C ratio from the calcium carbonate of this fossilis 0.011237. Compared to PDB, most of the natural compounds display anegative delta value. In the above equation, ¹³C/¹²C refers to theisotopomers.

[0070] Using the breath test of this invention, IRMS is an examplemethod to diagnose type 2 and gestational diabetes, and for monitoringglycemic control of diabetes patients.

EXAMPLE 5 ¹³C Glucose Breath Test Results of Normal, GestationalDiabetes and Impaired Glucose Tolerance Patient

[0071] Example 4 describes a method to analyze breath samples of thisinvention. FIG. 1 shows the mean (±SD) Delta per mil over Baseline (DOB)of the normal population. Also shown are the DOB's of a gestationaldiabetic and impaired glucose tolerance patients. Breath samplescollected 0, 1, 1.5 and 2 hours according to the protocol were analyzedby IRMS. IRMS analysis of the collected breath samples can be performedon various instruments, including but not limited to the AP2003 andAP2002 (Analytical Precision Ltd), ABCA (POZ Europa) and the Breath MAT(Finnigan MAT). The DOB values of the gestational diabetes and theimpaired glucose tolerance patients are well below the DOB of the normalpopulation (FIG. 1). The impaired glucose tolerance diagnosis wasinitially determined by OGTT, the gestational diabetes screen was usedto confirm gestational diabetes.

[0072] Impaired glucose tolerance (IGT) refers to a condition in whichblood sugar levels are higher than normal, but are not high enough to beclassified as diabetes. IGT is a major risk factor for type 2 diabetes.IGT is present in about 11 percent of adults, or approximately 20million Americans. About 40-45 percent of persons age 65 years of age orolder have either type 2 diabetes or IGT. A person is currentlydiagnosed with IGT when the 2-hour glucose results from a glucosetolerance test are greater than 7.8 mmol/L, but less than 11.0 mmol/L. Awoman is diagnosed with gestational diabetes when she is pregnant andhas any two of the following: a fasting plasma glucose of more than 5.3mmol/L, a 1-hour glucose level of more than 10.6 mmol/L, a 2-hourglucose level of more than 8.9 mmol/L. However, as this method ofdiagnosis is invasive, the breath tests of the current invention is thepreferred diagnosis method. The ¹³C glucose breath test is sensitive,accurate and non-invasive.

EXAMPLE 6 ¹³C Glucose Breath Test Results of a Normal, Insulin Resistantand Diabetes Patient

[0073] In this example, both breath test and blood glucose levels weredone on a normal, diabetic and insulin resistant patient. FIG. 2 showsthe DOB of 0, 1, 1.5 and 2 hours breath samples of a normal subjectanalyzed by IRMS. The blood glucose level of this normal individual isalso displayed.

[0074]FIG. 3 illustrates the breath test and blood glucose levels of adiabetic patient. The DOB of the breath samples are significantly lowerthan the DOB of the normal individual (FIG. 2), the blood glucose levelsare typical of a diabetic patient.

[0075] In FIG. 4, the breath test and blood glucose levels of aninsulin-resistant patient are depicted. The DOB of these breath samplesare significantly lower than the normal DOB (FIG. 2), the blood glucoselevels are typical of an insulin-resistant patient.

[0076] These results demonstrate one preferred utility of the breathtest of the current invention to diagnose diabetes and insulinresistance. In another aspect of the invention, the areas between thebreath test and blood glucose test curves can be used to diagnosepatients with insulin resistant or diabetes and confirm glucosetolerance in normal individuals by the comparison of the areas to thedifferent groups of normal, diabetic and insulin resistant patients.

[0077]FIG. 5 illustrates the ¹³C glucose breath test results of a normalindividual, insulin resistant and diabetes patient. The DOB's of theinsulin resistant and diabetes patients is significantly lower than thatof the normal DOB results.

EXAMPLE 7 NDIRS Instrumentation

[0078] Breath test samples of the invention can also be analyzed usingNDIRS instrumentation. The course of the ¹³CO₂/¹²CO₂ ratio in breathallows for diagnosis of diabetes. NDIRS can be further used to diagnosetype 2 and gestational diabetes patients and for monitoring therapy ofdiabetes patients (glycemic control of these patients).

[0079] The metabolism of ¹³C labeled substrate leads to a differentisotope ratio. NDIRS analysis of the invention can be performed onvarious instruments, including but not limited to the MicroLyzer(QuinTron), UbiT-IR200 and UbiT-100 (Otsuka Pharmaceutical Co., Ltd.),the URAS 10 (Hartmann and Braun) and the Isomax 2000 (Isotechnika).

EXAMPLE 8 Hyperinsulinemic Euglycemic Clamp Method for the Measurementof Insulin Resistance

[0080] Insulin resistance is defined as the decrease of the biologicalaction of insulin, and it mainly presents as an hyperinsulinemia. Thehyperinsulinemic euglycemic clamp is currently the reference method forquantifying insulin resistance. The clamp technique consists of infusinginsulin at a constant rate and, to prevent any decrease in the plasmaglucose level, by infusing dextrose. The rate of dextrose infused tomaintain euglycemia is an estimate of the amount of glucose, which istaken up by the tissues under the effect of a defined plasma insulinconcentration. Using several rates of insulin infusion allows theestablishment of the relationship between the whole body glucosedisposal and plasma insulin levels, and to discriminate between thestates of decreased insulin sensitivity and/or altered maximal capacityto dispose of glucose. However, the hyperinsulinemic euglycemic clampmethod is very invasive, time consuming, costly and variable. The breathtest of this invention is a preferred method to measure insulinresistance as it is reliable, sensitive, specific, cost-effective andnon-invasive.

EXAMPLE 9 Monitoring Long-Term Control of Diabetes

[0081] Measuring glycated hemoglobin is a current test used formonitoring long-term control of diabetes. Glycated hemoglobins areincreased as a reflection of hyperglycemia during the lifespan oferythrocytes. However, different analytical methods may measuredifferent glycated hemoglobins and caution must be exercised in theinterpretation of results. HPLC or column chromatography methods used toanalyse glycated hemoglobin are also highly sensitive to variations intemperature and pH. This test is also invasive, requiring several bloodsamples. The breath test of the present invention is preferred as it isnon-invasive, sensitive, accurate and cost-effective.

EXAMPLE 10 Usefulness of ¹³C Glucose Breath Test in Diagnosis ofDiabetes

[0082] Diabetes mellitus is a group of diseases characterized by highlevels of blood glucose resulting from defects in insulin secretion,insulin action, or both. Diabetes can be associated with seriouscomplications and premature death if left undiagnosed and untreated. Ithas been estimated by the World Health Organization that the number ofpeople suffering from diabetes worldwide will more than double fromabout 135 million now to 300 million by the year 2025. Of thoseestimated to have diabetes, it is believed that approximately one thirdof those are undiagnosed. It is also known that the prevalence ofdiabetes increases with age. It is estimated that 0.16% of people underthe age of 20 have diabetes but this number dramatically increases to18.4% for people over the age of 65.

[0083] There are four types of diabetes; type 1 (insulin dependent)represents 5 to 10% of all diagnosed cases, type 2(non-insulin-dependent diabetes) represents 90 to 95% of all diagnosedcases, gestational diabetes develops in 2 to 5% of all pregnancies butdisappears when a pregnancy is over, and other specific types ofdiabetes resulting from specific genetic syndromes, surgery, drugs,malnutrition, infections and other illnesses may account for 1 to 2% ofall diagnosed cases. A number of different methods exist for determiningdiabetes. These include postprandial blood glucose, oral glucosetolerance test (OGTT), O'Sullivan glucose tolerance test (gestationaltest), hemoglobin Alc, islet cell antibodies, glutamic aciddecarboxylase (GAD) antibodies, and insulin antibodies. However,diabetes is most readily detected when the carbohydrate metaboliccapacity is tested. This is done by stressing the system with a definedglucose load as in the OGTT.

[0084] Although the OGTT is a standard test for diabetes, it has beencriticized because many of the variables affecting the test results aredifficult to control for; the standardized carbohydrate diet, eight tosixteen hour fast, stress, exercise, hormone imbalances, and variousdrugs can cause test variables. These variables lead to poorreproducibility and limit the diagnostic usefulness of this test. Inaddition, the OGTT involves the collection of numerous blood specimensmaking it an invasive procedure.

[0085] The development of a ¹³C-glucose breath test for the detection ofdiabetes offers a non-invasive method that is not affected by the abovementioned variables. ¹³C is a non-radioactive isotope that occursnaturally in food and animal tissues. In the past the disadvantage of¹³C had been the shortage of the gas isotope mass spectrometers used foranalysis. With the ready availability of the necessary instrumentationand the ¹³C-labeled compounds required, the use of ¹³C-labeled compoundsin breath tests is more feasible.

[0086] Clinical Study

[0087] Objective: The primary aim of this pilot study is to evaluate thesensitivity, specificity and reliability of a ¹³C-D-glucose breath testin the diagnosis of type 2 and gestational diabetes as compared to thealready validated glucose tolerance test that will be considered thestandard.

[0088] Design: A multi-center, blinded, non-randomized design isutilized. Only the referring physicians have knowledge of theparticipants' status. Participants undergo a glucose tolerance test.Within two weeks following, participants undergo a ¹³C-D-glucose breathtest. The findings from both tests are examined for concordance.

[0089] STUDY PARTICIPANTS: This investigation is carried out byrecruiting 50 individuals each for type 2 and gestational diabetes. Fortype 2 diabetes, the participants are suspected to be diabetic. Forgestational diabetes, the participants are women in their 24th to 28thweek of pregnancy who have presented for the standard gestationaldiabetes mellitus screening test. Any diagnosis of diabetes is based onthe results of the glucose tolerance test.

[0090] TESTING STRATEGY: Eligible participants, after giving informedconsent, undergo the glucose tolerance test and the 13C-D-glucose breathtest separated by a minimum of 24 hours and a maximum of two weeks. Theglucose tolerance test is performed according to the guidelines of theCanadian Diabetes Association (CMAJ, JAMC Oct. 20, 1998;159(8suppl):S1-S29). Briefly, for the gestational diabetes screen, theglucose tolerance test consists of the consumption of a 50 g glucosetolerance drink and the collection of a venous blood sample one hourlater for glucose determination. For the time between the drinkconsumption and the blood sampling, the participant remains sedentaryand refrains from smoking or eating. Small sips of water may be taken ifnecessary.

[0091] For type 2 diabetes, an overnight fast (10-16 hours) precedes theglucose tolerance test. A fasting glucose blood sample is drawn prior tothe consumption of a 75 g glucose tolerance drink. Two hours after theingestion of the drink, a venous blood sample is collected for glucosedetermination. For the time between the drink consumption and the bloodsampling, the participant remains sedentary and refrains from smoking oreating. Small sips of water may be taken if necessary.

[0092] The ¹³C-D-glucose breath test is preceded by an overnight fast(minimum eight hours). After fasting, the participants are required toprovide a baseline breath sample. The participants then ingest the¹³C-D-glucose drink preparation and will provide breath samples at 1,1.5, and 2 hours. During the test the participants remain sedentary andare not permitted to smoke or eat. Only small sips of water arepermitted during the test.

[0093] OVERALL STUDY DESIGN: A total of 50 participants are investigatedeach for type 2 and gestational diabetes.

[0094] Visit One: During the recruitment process, each individual isasked to review a Participant Information Sheet and to talk with thelaboratory personnel to ensure that all eligibility requirements aremet. The individual is given an opportunity to ask questions and if theymeet all the eligibility criteria, they are asked to read and sign aninformed Consent Form.

[0095] All participants who have met the eligibility criteria and signeda consent form are tested by both the glucose tolerance test (Visit Two)and ¹³C-D-glucose breath test (Visit Three) separated by a minimum of 24hours and a maximum of two weeks.

[0096] Visit Two: The glucose tolerance test follows the guidelines setout by the Canadian Diabetes Association (CMAJ, JAMC Oct. 20, 1998;159(8suppl):S1-S29). Briefly, for the gestational diabetes screen, theparticipants are asked to consume a commercially available glucosetolerance drink consisting of 50 g of dextrose in 296 mL. One hourfollowing consumption, a venous blood sample is collected into ared-topped vacutainer tube. For type 2 diabetes, participants firstcomplete an overnight fast (10-16 hours) and then provide a fastingblood glucose sample. Participants then ingest a commercially availableglucose tolerance drink consisting of 75 g of dextrose in 296 mLfollowed by the collection of a venous blood sample 2 hourspost-consumption.

[0097] Visit Three: For the ¹³C-D-glucose breath test, participantsfirst complete an overnight fast (minimum of 8 hours). Participantsprovide a baseline breath sample which is followed by consumption of a¹³C-D-glucose-enriched solution containing 25 mg of ¹³C-D-glucose incombination with 15 g of unlabeled USP dextrose in 100 ml of water.Participants then provide breath samples at 1, 1.5, and 2 hours.

[0098] Note: Visit One and Visit Two may be combined if it is moreconvenient and all the testing criteria are met.

[0099] NUMBER OF PARTICIPANTS AND TARGET POPULATION: A total of 100adult participants (18 years of age or older) who are suspected ofhaving type 2 diabetes (n=50) or are being screened for gestationaldiabetes (n=50) are recruited from those individuals presenting for theoral glucose tolerance test.

[0100] INTERIM ANALYSIS: After 25 participants are enrolled for aparticular type of diabetes, all parties are unblinded to theparticipants' status. At this point in the study, the results areevaluated. If the ¹³C-D-glucose breath test results do not correlatewith the standard, the oral glucose tolerance test, such that greaterthan 5% of the participants are reported as false negatives or falsepositives, the study is temporarily halted. If the study is halted, theprotocol is amended to reflect an adjustment in the ¹³C-D-glucose breathtest kit components such that it contains 50 mg of ¹³C-D-glucose and 15g of unlabeled USP dextrose.

EXAMPLE 11 Advantages of the ¹³C Glucose Test for the Diagnosis ofDiabetes

[0101] The disadvantages of the OGTT include uncontrollable factorswhich cause variability or spurious results and the invasiveness of thetest. Other tests known in the art are not specific, are invasive, arevariable and are labor intensive. The ¹³C glucose breath test of thepresent invention is sensitive, reliable and specific. The ¹³C glucosebreath test shows minimal intra-individual variation, excellentanalytical precision and breath specimens are stable for at least sixweeks at room temperature. The ¹³C glucose breath test is preferred overtests known in the art, it is non-invasive, easy to perform, has verygood sensitivity and specificity and is cost effective. A preferred useof the breath test of this invention is for the diagnosis of type 2 andgestational diabetes. This invention is also preferred to determine thelevel of insulin resistance and for monitoring the appropriateness ofthe therapy of diabetes patients.

[0102] Further variations and modification of the present invention willbe apparent to those skilled in the art and are intended to beencompassed by the specification and claims appended hereto.

We claim:
 1. A diagnostic kit for the determination of glycemic controlin a subject comprising: a predetermined quantity of ¹³C-enrichedglucose and a breath collection container.
 2. A diagnostic kit accordingto claim 1 further comprising a plurality of breath collectioncontainers.
 3. A diagnostic kit according to claims 1 or 2 for use inthe diagnosis of diabetes.
 4. A diagnostic kit according to claims 1 or2 for use in the diagnosis of insulin resistance.
 5. A diagnostic kitaccording to claims 1 or 2 for use in the diagnosis of gestationaldiabetes.
 6. A diagnostic kit according to claims 1 or 2 for use in thedetermination of adequacy of antihyperglycemic therapy.
 7. A diagnostickit according to claims 1-6 wherein the ¹³C-enriched glucose isuniformly enriched.
 8. A diagnostic kit according to claims 1-6, furthercomprising a tube that is adapted for the transfer of the breath of asubject into the breath collection container.
 9. A diagnostic kitaccording to claims 1-8 further comprising a set of instructions whereinthe instructions direct the subject to collect a first breath sample ina first breath collection container, ingest the ¹³C-enriched glucose andcollect a second breath sample at a time point that is about 90 minutesafter ingestion of the ¹³C-enriched glucose.
 10. The use of ¹³C-enrichedglucose for the determination of glycemic control in a subject. 11.¹³C-Enriched glucose for use in the manufacture of a diagnostic kit forthe determination of glycemic control in a subject.
 12. The ¹³C-enrichedglucose of claim 11 for use in the manufacture of a diagnostic kit forthe detection of diabetes.
 13. The ¹³C-enriched glucose of claim 11 foruse in the manufacture of a diagnostic kit for the detection ofgestational diabetes.
 14. The ¹³C-enriched glucose of claim 11 for usein the manufacture of a diagnostic kit for the detection of insulinresistance.
 15. The ¹³C-enriched glucose of claim 11 for use in themanufacture of a diagnostic kit for the determination of adequacy ofantihyperglycemic therapy.
 16. A diagnostic kit for the determination ofglycemic control in normal, diabetic and insulin resistant subjects bycomparing blood glucose levels with breath levels of ¹³C-enriched CO₂comprising: a predetermined quantity of ¹³C-enriched glucose, a breathcollection container and a blood sampling device.